Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The present invention relates to novel compositions and methods, for incorporating amino acid analogues into proteins in vivo, by overexpression of aminoacyl-tRNA synthetases.
Expanding the scope of biological polymerizations to include non-natural monomers, is an area of growing interest, with important theoretical and practical consequences. An early and critically important example of such studies was the demonstration that xe2x80x9cdideoxyxe2x80x9d nucleotide monomers can serve as substrates for DNA polymerases. Advances in DNA sequencing (F. Sanger, S. Nicklen, A. R. Coulson, Proc. Natl. Acad. Sci. USA 1977, 74, 5463-5467), DNA base pairing models (M. J. Lutz, S. A. Benner, S. Hein, G. Breipohl, E. Uhlmann, J. Am. Chem. Soc. 1997, 119, 3177-3178; J. C. Morales, E. T. Kool, Nature Struct. Biol. 1998, 5, 950-954), materials synthesis (W. H. Park, R. W. Lenz, S. Goodwin, Macromolecules 1998, 31, 1480-1486; Y. Doi, S. Kitamura, H. Abe, Macromolecules 1995, 28, 4822-4828), and cell surface engineering (K. J. Yarema, L. K. Mahal, R. E. Bruehl, E. C. Rodriguez, C. R. Bertozzi, J. Biol. Chem. 1998, 273, 31168-31179; L. K. Mahal, K. J. Yarema, C. R. Bertozzi, Science 1997, 276, 1125-1128; Saxon, E. and Bertozzi, C. R. Science 2000, 287, 2007-2010) have resulted from the recognition of non-natural monomers by the enzymes that control these polymerizations.
Recent investigations have shown the incorporation of modified or completely xe2x80x9csyntheticxe2x80x9d bases into nucleic acids (Matray, T. J.; Kool, E. T. Nature 1999, 399, 704; Kool, E. T. Biopolymers 1998, 48, 3; Morales, J. C. ; Kool, E. T. Nature Struct. Biol. 1998, 5, 950; Guckian, K. M. ; Kool, E. T. ; Angew. Chem. Int. Ed. Eng. 1998, 36, 2825; Liu, D. Y. ; Moran, S.; Kool, E. T. Chem. Biol. 1997, 4, 919; Moran, S.; Ren, R. X. F.; Kool, E. T. Proc. Natl. Acad. Sci. USA 1997, 94, 10506; Moran, S. et al. J. Am. Chem. Soc. 1997, 119, 2056; Benner, S. A. et al. Pure Appl. Chem. 1998, 70, 263; Lutz, M. J.; Horlacher J.; Benner, S. A. Bioorg. Med. Chem. Lett. 1998, 8, 1149; Lutz, M. J. ; Held, H. A. ; Hottiger, M.; Hubscher, U.; Benner, S. A. Nuc. Acids Res. 1996, 24, 1308; Horlacher, J. et al. Proc. Natl. Acad. Sci. USA 1995, 92, 6329; Switzer, C. Y. ; Moroney, S. E. ; Benner, S. A. Biochemistry 1993, 32, 10489; Lutz, M. J.; Horlacher, J.; Benner, S. A. Bioorg. Med. Chem. Lett. 1998, 8, 499; Switzer, C.; Moroney, S. E. ; Benner, S. A. J. Am. Chem. Soc. 1989, 111, 8322; Piccirilli, J. A. ; Krauch, T.; Moroney, S. E. ; Benner, S. A. Nature 1990, 343, 33), while materials researchers have exploited the broad substrate range of the poly(xcex2-hydroxyalkanoate) (PHA) synthases to prepare novel poly(xcex2-hydroxyalkanoate)s (PHAs) with unusual physical properties (Kim, Y. B.; Rhee, Y. H.; Lenz, R. W. Polym. J. 1997, 29, 894; Hazer, B.; Lenz, R. W.; Fuller, R. C. Polymer 1996, 37, 5951; Lenz, R. W. ; Kim, Y. B. ; Fuller, R. C. FEMS Microbiol. Rev. 1992, 103, 207; Park, W. H.; Lenz, R. W.; Goodwin, S. Macromolecules 1998, 31, 1480; Ballistreri, A. et al. Macromolecules 1995, 28, 3664; Doi, Y.; Kitamura, S.; Abe, H. Macromolecules 1995, 28, 4822).
Novel polymeric materials with unusual physical and/or chemical properties are also useful in polymer chemistry. The last several decades have shown many advances in synthetic polymer chemistry that provide the polymer chemist with increasing control over the structure of macromolecules (Szwarc, M. Nature 1956, 178, 1168-1169 Szwarc, M. Nature 1956, 178, 1168-1169; Faust, R.; Kennedy, J. P. Polym. Bull. 1986, 15, 317-323; Schrock, R. R. Acc. Chem. Res. 1990, 23, 158-165; Corradini, P. Macromol Symp. 1995, 89, 1-11; Brintzinger, H. H.; Fischer, D.; Mulhaupt, R.; Rieger, B.; Waymouth, R. M. Angew. Chem. Int. Ed. Engl. 1995, 34, 1143-1170; Dias, E. L.; SonBinh, T. N.; Grubbs, R. H. J. Am. Chem. Soc. 1997, 119, 3887-3897; Chiefari, J. et al. Macromolecules 1998, 31, 5559-5562). However, none of these methods have provided the level of control that is the basis of the exquisite catalytic, informational, and signal transduction capabilities of proteins and nucleic acids (Ibba, M.; Soll, D. Science 1999, 286, 1893-1897). There remains a need for control over protein synthesis to design and produce artificial proteins having advantageous properties.
For this reason, the design and synthesis of artificial proteins that exhibit novel and potentially useful structural properties have been investigated. Harnessing the molecular weight and sequence control provided by in vivo synthesis would permit control of folding, functional group placement, and self-assembly at the angstrom length scale. Proteins that have been produced by in vivo methods exhibit predictable chain-folded lamellar architectures (Krejchi, M. T.; Atkins, E. D. T.; Waddon, A. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Science 1994, 265, 1427-1432; Parkhe, A. D.; Fournier, M. J. ; Mason, T. L.; Tirrell, D. A. Macromolecules 1993, 26(24), 6691-6693; McGrath, K. P.; Fournier, M. J. ; Mason, T. L.; Tirrell, D. A. J. Am. Chem. Soc. 1992, 114, 727-733; Creel, H. S.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Macromolecules 1991, 24, 1213-1214), unique smectic liquid-crystalline structures with precise layer spacings (Yu, S. M.; Conticello, V.; Zhang, G.; Kayser, C.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Nature 1997, 389, 187-190), and controlled reversible gelation (Petka, W. A.; Hardin, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Science 1998, 281, 389-392). The demonstrated ability of these protein polymers to form unique macromolecular architectures will be of importance for engineering materials with interesting liquid-crystalline, crystalline, surface, electronic, and optical properties.
Novel chemical and physical properties that can be engineered into protein polymers may be expanded by the precise placement of amino acid analogues. Efforts to incorporate novel amino acids into proteins in vivo have relied on the ability of the translational apparatus to recognize amino acid analogues that differ in structure and functionality from the natural amino acids. The in vivo incorporation of amino acid analogues into proteins is controlled most stringently by the aminoacyl-tRNA synthetases (AARS), the class of enzymes that safeguards the fidelity of amino acid incorporation into proteins (FIG. 1). The DNA message is translated into an amino acid sequence via the pairing of the codon of the messenger RNA (mRNA) with the complementary anticodon of the aminoacyl-tRNA. Aminoacyl-tRNA synthetases control the fidelity of amino acid attachment to the tRNA. The discriminatory power of the aminoacyl-tRNA synthetase places severe limits on the set of amino acid structures that can be exploited in the engineering of natural and artificial proteins in vivo.
Several strategies for circumventing the specificity of the synthetases have been explored. Introduction of amino acid analogues can be achieved relatively simply via solid-phase peptide synthesis (Merrifield, R. B. Pure and Appl. Chem. 1978, 50, 643-653). While this method circumvents all biosynthetic machinery, the multistep procedure is limited to synthesis of peptides less than or equal to approximately 50 amino acids in length, and is therefore not suitable for producing protein materials of longer amino acid sequences.
Chemical aminoacylation methods, introduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992, 25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M. Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. J. Biol. Chem. 1978, 253, 4517) and exploited by Schultz, Chamberlin, Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989, 244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J. Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356, 537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997, 4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W. et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996, 271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), avoid the synthetases altogether, but provide low protein yields.
Alteration of the synthetase activities of the cell is also possible, either through mutagenesis or through introduction of heterologous synthetases (Ibba, M.; Hennecke, H. FEBS Lett. 1995, 364, 272; Liu, D. R.; Maghery, T. J.; Pastrnak, M.; Schultz, P. G. Proc. Natl. Acad. Sci. USA, 1997, 94, 10092; Furter, R. Protein Sci. 1998, 7, 419; Ohno, S. et al., J. Biochem. 1998, 124, 1065; Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. 1999, 96, 4780; Wang, L.; Magliery, T. J.; Liu, D. R.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 5010-5011; Pastrnak, M.; Magliery, T. J.; Schultz, P. G. Helv. Chim. Acta 2000, 83, 2277-2286).
In some instances, the ability of the wild-type synthetases to accept amino acid analogues has been exploited. For example, wild-type synthetases have been shown to activate and charge substrates other than the canonical, proteinogenic amino acids (Cowie, D. B.; Cohen, G. N. Biochim. Biophys. Acta. 1957, 26, 252; Richmond, M. H. Bacteriol Rev. 1962, 26, 398; Horton, G.; Boime, I. Methods Enzymol. 1983, 96, 777; Wilson, M. J.; Hatfield, D. L. Biochim. Biophys. Acta 1984, 781, 205). This approach offers important advantages with respect to synthetic efficiency, in that neither chemical acylation of tRNA nor cell-free translation is required. The simplicity of the in vivo approach, its relatively high synthetic efficiency, and its capacity for multisite substitution, make it the method of choice for production of protein materials whenever possible.
The capacity of the wild-type translational apparatus has been previously demonstrated to utilize amino acid analogues bearing fluorinated (Richmond, M. H. J. Mol. Biol. 1963, 6, 284; Fenster, E. D.; Anker, H. S. Biochemistry 1969, 8, 268; Yoshikawa, E.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Macromolecules 1994, 27, 5471), unsaturated (Van Hest, J. C. M.; Tirrell, D. A. FEBS Lett. 1998, 428, 68; Deming, T. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. J. Macromol. Sci.xe2x80x94Pure Appl. Chem. 1997, A34, 2134), electroactive (Kothakota, S.; Mason, T. L.; Tirrell, D. A.; Fournier, M. J. J. Am. Chem. Soc. 1995, 117, 536), and other useful side chain functions. The chemistries of the above functional groups are distinct from the chemistries of the amine, hydroxyl, thiol, and carboxylic acid functional groups characteristic of proteins; this makes their incorporation particularly attractive for targeted chemical modification of proteins.
For example, alkene functionality introduced into artificial proteins via dehydroproline can be quantitatively modified via bromination and hydroxylation (Deming, T. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. J. Macromol. Sci. Pure Appl. Chem. 1997, A34, 2143-2150). Alkene functionality, introduced by incorporation of other amino acid analogues, should be useful for chemical modification of proteins by olefin metathesis (Clark, T. D.; Kobayashi, K.; Ghadiri, M. R. Chem. Eur. J. 1999, 5, 782-792; Blackwell, H. E.; Grubbs, R. H. Angew. Chem. Int. Ed. Engl. 1998, 37, 3281-3284), palladium-catalyzed coupling (Amatore, C.; Jutand, A. J. Organomet. Chem. 1999, 576, 255-277; Tsuji, J. Palladium Reagents and Catalysts. Innovations in Organic Synthesis; John Wiley and Sons: New York, 1995; Schoenberg, A.; Heck, R. F. J. Org. Chem. 1974, 39, 3327-3331), and other chemistries (Trost, B. M.; Fleming, I., Eds. Comprehensive Organic Synthesis; Pergamon Press: Oxford, 1991). The incorporation of fluorinated functional groups into proteins has imparted to protein films the low surface energy characteristic of fluoropolymers; contact angles of hexadecane on fluorinated protein polymers (70xc2x0) are much higher than those on unfluorinated controls (17xc2x0) (Yoshikawa, E.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Macromolecules 1994, 27, 5471-5475).
Methionine (1) (FIG. 1) is a possible target for substitution by amino acid analogues, with its hydrophobicity and polarizability, make it an important amino acid for regulating protein structure and proteinxe2x80x94protein recognition processes (T. Yuan, A. M. Weljie, H. J. Vogel, Biochemistry 1998, 37, 3187-3195; H. L. Schenck, G. P. Dado, S. H. Gellman, J. Am. Chem. Soc. 1996, 118, 12487-12494; Maier, K. L.; Lenz, A. G., Beck-Speier, I.; Costabel, U. Methods Enzymol. 1995, 251, 455-461). Replacement of methionine by its analogues may therefore permit purposeful manipulation of these properties.
Several analogues of methionine (1), specifically selenomethionine, telluromethionine, norleucine, trifluoromethionine and ethionine (Hendrickson, W. A.; Horton, J. R.; Lemaster, D. M. EMBO J. 1990, 9, 1665; Boles, J. O. et al Nature Struct. Biol. 1994, 1, 283; Cowie, D. B.; Cohen, G. N.; Bolton, E. T.; de Robichon-Szulmajster, H. Biochim. Biophys. Acta 1959, 34, 39; Duewel, H.; Daub, E.; Robinson, R.; Honek, J. F. Biochemistry 1997, 36, 3404; Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn, C.; Kellerman, J.; Huber, R. Eur. J. Biochem. 1995, 230, 788), have been shown to exhibit translational activity in bacterial hosts. Incorporation of selenomethionine in place of methionine has long been known to facilitate protein structure determination by x-ray crystallography (Wei, Y.; Hendrickson, W. A.; Crouch, R. J.; Satow, Y. Science 1990, 249, 1398-1405).
However, only a limited number of amino acid analogues have been shown to conclusively exhibit translational activity in vivo, and the range of chemical functionality accessible via this route remains modest. These circumstances dictate a need for a systematic search for new amino acid analogues and strategies that will allow the engineering of proteins with novel chemical and physical properties.
The present invention seeks to overcome these and other disadvantages in the prior art by providing a novel method for incorporating amino acid analogues into polypeptides of interest in vivo by expanding the scope of amino acid analogues that are incorporated and increasing protein yields. Preferably, the production of modified polypeptides can be in a host-vector system in which a natural amino acid in the wild-type polypeptide is replaced with a selected amino acid analogue by overexpressing an aminoacyl-tRNA synthetase corresponding to the natural amino acid so replaced.
In addition, the present invention provides novel host-vector systems. The host-vector system produces an aminoacyl-tRNA synthetase in an amount in excess of the level of a naturally occurring aminoacyl-tRNA synthetase. The system also produces a polypeptide of interest in an amount in excess of the level produced by a naturally occurring gene encoding the polypeptide of interest.
Nucleic acids encoding the expression vectors, hosts, and methods of integrating a desired amino acid analogue into target polypeptides are also provided.
The invention further provides purified dihydrofolate reductase polypeptides, produced by the methods of the invention, in which the methionine residues have been replaced with homopropargylglycine (2-amino-hexynoic acid), homoallylglycine (2-amino-hexenoic acid), cis-crotylglycine (cis-2-amino-4-hexenoic acid), trans-crotylglycine (trans-2-amino-4-hexenoic acid), norleucine, 6,6,6-trifluoro-2-amino hexanoic acid, 2 -amino-heptanoic acid, norvaline, o-allylserine, 2-butynylglycine, allylglycine or propargylglycine. The formation of the modified polypeptides demonstrate the ease and efficiency of the methods of the invention for incorporating amino acid analogues such as, methionine analogues, into proteins such as, dihydrofolate reductase.
Using the methods of the invention, it is possible to produce entirely new polypeptides containing amino acid analogues having unusual properties.